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Background:CTNNB1 is reported to be related to the pathological process of ischemic stroke (IS) and coronary artery disease (CAD). Polymorphism located in the 3′ untranslated region (3′UTR) of a gene might affect gene expression by modifying binding sites for microRNAs (miRNAs). This study aimed to analyze the association between polymorphism rs2953, which locates in the 3′UTR of CTNNB1, and the risk of IS and CAD. Methods: The CTNNB1 messenger RNA (mRNA) expression level in peripheral venous blood was measured. In total, 533 patients with IS, 500 patients with CAD, and 531 healthy individuals were genotyped by Sequenom Mass-Array technology. The binding of miR-3161 to CTNNB1 was determined by dual-luciferase reporter assay. Results: The CTNNB1 mRNA expression level for the IS group was significantly lower than that for the control group. Rs2953 was significantly associated with both IS risk and CAD risk. Significant association was also found between polymorphism rs2953 and many conventional factors, such as serum lipid level, blood coagulation markers, blood glucose level, and homocysteine level in patients. Rs2953 T allele introduced a binding site to miRNA-3161 and thus decreased luciferase activity. Conclusion: Polymorphism rs2953 is associated with the risk of both IS and CAD. Moreover, polymorphism rs2953 (T) introduces a binding site to miRNA-3161 and thus decreases luciferase activity in cell lines. [ABSTRACT FROM AUTHOR]